Measuring Single-Cell Mitochondrial DNA Copy Number and Heteroplasmy Using Digital Droplet Polymerase Chain Reaction

Summary

Here we present a protocol for measuring absolute mitochondrial (mt)DNA copy number and mtDNA deletion heteroplasmy levels in single cells.

Abstract

The mammalian mitochondrial (mt)DNA is a small, circular, double-stranded, intra-mitochondrial DNA molecule, encoding 13 subunits of the electron transport chain. Unlike the diploid nuclear genome, most cells contain many more copies of mtDNA, ranging from less than 100 to over 200,000 copies depending on cell type. MtDNA copy number is increasingly used as a biomarker for a number of age-related degenerative conditions and diseases, and thus, accurate measurement of the mtDNA copy number is becoming a key tool in both research and diagnostic settings. Mutations in the mtDNA, often occurring as single nucleotide polymorphisms (SNPs) or deletions, can either exist in all copies of the mtDNA within the cell (termed homoplasmy) or as a mixture of mutated and WT mtDNA copies (termed heteroplasmy). Heteroplasmic mtDNA mutations are a major cause of clinical mitochondrial pathology, either in rare diseases or in a growing number of common late-onset diseases such as Parkinson's disease. Determining the level of heteroplasmy present in cells is a critical step in the diagnosis of rare mitochondrial diseases and in research aimed at understanding common late-onset disorders where mitochondria may play a role. MtDNA copy number and heteroplasmy have traditionally been measured by quantitative (q)PCR-based assays or deep sequencing. However, the recent introduction of ddPCR technology has provided an alternative method for measuring both parameters. It offers several advantages over existing methods, including the ability to measure absolute mtDNA copy number and sufficient sensitivity to make accurate measurements from single cells even at low copy numbers. Presented here is a detailed protocol describing the measurement of mtDNA copy number in single cells using ddPCR, referred to as droplet generation PCR henceforth, with the option for simultaneous measurement of heteroplasmy in cells with mtDNA deletions. The possibility of expanding this method to measure heteroplasmy in cells with mtDNA SNPs is also discussed.

Introduction

Mammalian mitochondrial (mt)DNA is a small (approx. 16.5 Kb), circular DNA genome residing in the mitochondrial matrix that encodes 37 genes, comprising two rRNAs, 22 tRNAs, and 13 protein-coding genes¹. Unlike the nuclear genome, which contains one (haploid) or two (diploid) copies of each gene per cell, mtDNA is present in multiple copies in the mitochondria of each cell, ranging from tens of copies (e.g., mature spermatocytes) to hundreds of thousands of copies (e.g., oocytes)^2,3. A consequence of this multi-copy nature is that mutations in the mtDNA genome, which may exist as single-nucleotide polymorphisms (SNPs), deletions, or duplications, can be present at varying levels in any given cell, making up anywhere from 0% to 100% of the cell's total mtDNA population. The existence of wild-type and mutant mtDNA genomes in the same cell is termed heteroplasmy, and pathogenic heteroplasmic mtDNA mutations are a major cause of mitochondrial disease, with several common neurological syndromes linked to underlying heteroplasmic mtDNA mutations⁴.

Two key parameters that contribute to the likelihood of a heteroplasmic mtDNA mutation causing clinical disease are the heteroplasmy level and the mtDNA copy number. Many heteroplasmic mutations display a threshold effect, with biochemical and clinical phenotypes only becoming apparent above a certain heteroplasmy level, typically around 80%⁵, and subsequently worsening as heteroplasmy increases further⁴. However, it is also important to consider the number of copies of mtDNA present in the cell, as this will influence the number of wild-type (i.e., 'healthy') mtDNA genomes that are present at a given heteroplasmy level. Studies in mitochondrial disease patients have highlighted the importance of this interplay between heteroplasmy and copy number^5,6, and Filograna et al. recently reported an increase in mtDNA copy number alleviating symptoms despite unchanged heteroplasmy in a mouse model of mitochondrial disease⁷.

Whilst much has been done in recent years to improve the understanding of the pathogenesis and transmission of diseases caused by mtDNA heteroplasmy, most of this work has been conducted at the level of tissues rather than cells, comparing mean tissue heteroplasmy levels and copy number measurements acquired from bulk tissue biopsies and blood samples. Some well-established techniques, such as laser capture microdissection, allow for such measurements at the cellular level^8,9; however, the recent explosion of high-throughput single-cell analysis methods, or so-called "Single-cell Omics"¹⁰, has created a requirement for methods that can accurately measure these crucial mtDNA parameters at the single-cell level.

The droplet generation PCR method takes advantage of recent advances in microfluidic technology to improve upon existing methods of quantitating unknown DNA concentrations via PCR amplification of specific target amplicons in the sample DNA¹¹. Unlike qPCR, where the sample DNA is amplified in a single reaction, with the relative rate of accumulation of PCR product acting as the readout, this method splits the initial sample into thousands of individual droplets, thus compartmentalizing the sample DNA molecules into spatially separate reactions¹². The subsequent PCR reaction proceeds in each individual droplet, with the PCR product only accumulating in droplets that contain the target DNA. The result of this reaction is a digital output in the form of a pool of droplets that either contain a copy of the target DNA and amplified PCR product, or do not contain target DNA. Using fluorescent DNA probes or a double-stranded (ds)DNA binding dye, the droplets containing amplified product can be counted, and the ratio of 'positive' to 'negative' droplets can be used to calculate the absolute number of DNA copies that were present in the initial sample. This absolute measurement, as opposed to the relative measurement acquired from a qPCR reaction, is the key factor that allows this methodology to be used for accurate measurement of mtDNA copy number and heteroplasmy in single cells¹¹, and several recent studies have already utilized droplet generation PCR technology for this purpose^13,14. This article presents a method for measuring mtDNA copy number and deletion heteroplasmy in single cells from both human and mouse tissue.

Protocol

All experiments followed the ARRIVE guidelines and were approved by the University of Cambridge Animal Welfare Ethical Review Body (AWERB).

NOTE: All sample preparation steps prior to droplet generation must be performed in a clean pre-PCR work area, ideally in a UV-sterilized cabinet where possible. The protocol described here uses specific droplet generation PCR equipment (see Table of Materials), and while the general method should be applicable to other systems, it is recommended to consult the manufacturer's guidelines regarding primer/probe concentrations, PCR cycling conditions, etc., as they may differ from those described here. Cell lines/primary cells used in this study were as follows: Human HeLa cells (commercially obtained), human HEK 293T cells (commercially obtained), primary human dermal fibroblast cells (obtained from the Newcastle Biobank), human cybrids (WT & ΔH2.1 deletion¹⁵, obtained from C. Moraes, University of Miami), mouse embryonic fibroblasts (immortalized, from C57Bl/6 mice, obtained from J. Stewart, Newcastle University), mouse primordial germ cells (obtained from C57Bl/6 mouse embryos), and mouse MII oocytes (obtained from adult female C57Bl/6 mice). All cultured cells were maintained in High Glucose (4.5g/L) DMEM supplemented with 10% fetal bovine serum at 37 °C with 5% CO₂. Primary mouse cells used in this study were isolated from animals kept according to the Animal (Scientific Procedures) Act 1986 under Home Office Project License P6C97520A.

1. Design and synthesize primer & probe sets targeting DNA sequences of interest

NOTE: Droplet generation PCR can also be performed using a dsDNA binding dye in place of the amplicon-specific probes.

1. Design the primer and probe sequences according to the guidelines given by the system manufacturer. Validated primer and probe sequences suitable for measuring single-cell mtDNA copy number/deletion heteroplasmy in both human^16,17 and mouse¹⁸ cells are provided in Table 1. When selecting alternative target sequences, consider the following points.
   1. Multiplex two primer/probe sets in a single PCR assay but ensure that the two assays utilize one FAM-labeled probe and one HEX-labeled probe so that the target amplicons can be differentiated by the droplet reader. This protocol presents a duplex probe assay. With careful experimental design, multiplexing can be increased up to four targets, and alternative platforms can achieve higher-dimensional multiplexing.
   2. When multiplexing primer/probe sets targeting separate mtDNA sequences, ensure that the amplicons generated by the two primer sets do not overlap.
   3. When measuring heteroplasmy in samples carrying mtDNA deletions, ensure that one target amplicon falls within the expected deleted region and the other falls outside of the expected deleted region.
      NOTE: Optimal PCR cycling conditions for alternative assays may differ from those quoted in Step 5.1; see the Discussion for further details on assay optimization.

2. Isolation of DNA from single cells

NOTE: This method can also be used for small bulk samples of up to 100 cells.

1. Collect single cells using an appropriate method, such as Fluorescence-Activated Cell Sorting (FACS)¹⁹ or laser capture microdissection²⁰, in as small a volume as possible into a suitable receptacle (e.g., 96-well plate). Use of a cell viability dye prior to or during single-cell isolation will minimize the likelihood of isolating dead cells. Sort the single cells directly into lysis buffer if desired and store at -80 °C (up to 6 months) prior to analysis.
2. Lyse the cells in a small volume (<10 µL) of a suitable lysis buffer (the buffer used in this study is described in step 2.2.1.).
   NOTE: All data obtained from cells in this study are from single-cell lysates or pools of 20 cells (sample sizes are stipulated in the figure legends). Efficient formation of an oil/droplet emulsion during the droplet generation step of the droplet generation PCR protocol can be significantly affected by the presence of detergents in the sample; therefore, it is recommended to validate the compatibility of all cell-lysis buffers with droplet generation at the intended input concentration before proceeding with experimental samples and using the smallest practical volume of lysis buffer. The following lysis protocol results in minimal impact on droplet generation efficiency.
   1. Prepare the lysis buffer containing 50 mM Tris-HCl pH 8.3, 1% TWEEN-20, and 200 µg/mL Proteinase K.
   2. Add 2.5 µL of the lysis buffer to each sample, seal the plate with an adhesive plate seal, and then centrifuge the samples at 1,000 x g for 1 min at 4 °C.
   3. Incubate the samples at 37 °C for 30 min on the thermocycler with the heated lid set at 105 °C to prevent the condensation of liquid on the plate cover.
   4. Add 7.5 µL of nuclease free water to each sample to get a final sample volume of 10 µL, re-seal the plate, and then centrifuge the samples at 1,000 x g for 1 min at 4 °C.
   5. Incubate on the thermocycler at 80 °C for 15 min (heated lid set to 105 °C) to inactivate Proteinase K.
   6. Centrifuge the samples at 1,000 x g for 1 min at 4 °C and then keep on ice.
3. Proceed to step 3 below.
   NOTE: Cell lysates can be stored at -20 °C prior to continuing with step 3; however, use freshly eluted DNA where possible to eliminate the risk of freeze-thaw cycles resulting in DNA degradation.

3. Preparation of samples

NOTE: Ensure that technical replicates of samples and non-template controls (NTCs) are included on each assay plate to ensure the accuracy of results. The dynamic range of the droplet generation PCR assay is up to 120,000 copies of the target amplicon per reaction. For single-cell or small bulk-cell sample lysates, dilution is unlikely to be necessary, and the lysate mixture can be input directly to the reaction for the measurement of mtDNA copy number (e.g., a lysate sample of 10 µL can be split and run in triplicate with 3 µL input directly into each assay). However, using undiluted lysate from cells with very high copy mtDNA numbers (e.g., oocytes) or containing larger numbers of cells (e.g., 50-100) may exceed this dynamic range. In such cases, an initial serial dilution is required to identify an appropriate dilution factor that avoids saturation of the assay for that specific cell type/cell number (see Representative Results for further details).

1. Defrost all reagents on ice, vortex, and spin down briefly before use.
2. Prepare the master mix (minus sample DNA) on ice as described in (Table 2).
   NOTE: If only one target amplicon is being measured, add an additional 2.55 µL of nuclease-free water per sample to achieve the final volume of 22 µL. Prepare sufficient master mix for the number of samples and NTCs to be analyzed, plus enough to account for any 'blank' wells required on the droplet generation PCR plate (see next step).
3. Vortex the prepared master mix briefly to mix and aliquot the required volume (22 µL minus input DNA volume) into each well of a droplet generation PCR 96-well plate. Arrange the samples in full columns on the 96-well plate; fill any empty wells in incomplete columns with master mix and use them as NTCs.
4. Add the input DNA to each sample well and nuclease-free water/elution buffer to NTC wells to bring the total volume of each up to 22 µL.
5. Seal the plate with an adhesive plate seal and place on a shaker at 2,000 rpm for 1 min, and then centrifuge at 1,000 x g for one min at 4 °C.
6. Place the plate on ice and proceed to Step 4.
   NOTE: Prepared plates can be stored on ice and protected from light for short periods of time (e.g., 1-2 h) before proceeding with droplet generation.

4. Generation of droplets

NOTE: Refer to Figure 1A for the schematic of the droplet generator instrument deck referenced in this section.

1. Power on the droplet generator.
2. Check that a bottle of droplet generating oil is loaded to position E of the instrument deck. If prompted, follow the on-screen instructions to replace the bottle.
3. Click Configure Sample Plate on the touch screen. Select all columns on the sample plate that contain samples/blanks.
   NOTE: Entry of plate name and details of the experiment at this step is optional and not required to continue setting up the experiment.
4. Click OK to confirm the plate configuration; orange lights will illuminate on the instrument deck to indicate positions where consumables need to be loaded.
5. Load the droplet generator cartridges to position B on the instrument deck so that all lights turn green.
6. Place an empty tip waste trough into position C on the instrument deck.
7. Remove lids from filter tip boxes and load to position D on the instrument stage so that all lights turn green.
8. Remove the adhesive plate seal from the sample plate and load to position F of the instrument deck so that the light turns green.
9. Place an empty droplet collection 96-well plate into a cool block (pre-chilled at -20 °C) and load to position G of the instrument deck so that the light turns green.
10. Click Start Droplet Generation on the instrument touch screen.
11. Click Start Run on the instrument touch screen. The instrument lid will close automatically. After initializing, the time remaining until droplet generation is complete will display on the instrument touch screen.
12. Once the droplet generation is complete, check the sample collection plate visually to confirm that a droplet emulsion layer is present above the oil phase in each well (Figure 1B).
    NOTE: If a droplet emulsion layer is not visible, do not proceed further with the experiment and check for any errors that may have occurred during previous steps of the protocol.
13. Clear the used consumables from the instrument deck and empty the tip-waste trough.
14. Power on the plate sealer, set the temperature to 180 °C and seal time to 5 s, and allow the machine to reach the operating temperature.
15. Put the sample collection plate into the plate sealer plate-holder and place a fresh foil seal on top of the plate with the red line facing upwards. Take care not to touch the underside of the foil seal.
    NOTE: The plate holder should be stored at room temperature (RT) and only placed into the plate sealer drawer when a foil seal is being applied to prevent heat transfer to the sample collection plate.
16. When the plate sealer has reached operating temperature, press Eject on the instrument touchscreen; the drawer will open automatically.
17. Place the plate holder into the plate sealer drawer and press Seal. The drawer will close automatically and then open again once sealing is complete.
18. Replace the sealed plate on the cold block, turn off the plate sealer, and proceed immediately to Step 5.
    NOTE: At this stage, the droplets are unstable, so ensure that the PCR step is commenced within 1 h of droplet generation finishing.

5. PCR

1. Place the sealed droplet collection plate onto a PCR cycler equipped with a 96-deep-well block and run the thermal cycling protocol described in Table 3.
   NOTE: Set heated lid temperature to 105 °C and sample volume to 40 µL. Denaturation and annealing/extension steps must include a temperature ramp-rate of 2 °C/s to ensure that the oil has time to equilibrate to the correct temperature.
2. Proceed to Step 6.
   NOTE: Once the thermal cycling protocol is complete, the droplets are more stable, and the plate can be stored for up to 4 days at 4 °C before proceeding with the next step.

6. Droplet reading

1. Power on the droplet reader and connected computer.
2. Check the fluid levels in the droplet reader oil and droplet reader waste bottles and fill/empty these, respectively, if required.
3. Load the plate containing the post-PCR samples into the droplet reader plate holder, place the cover on top of the holder and secure it in place with the black locking clips. Do not remove the foil lid from the sample plate.
4. Press the Open/Close button on the lid of the droplet reader to open it.
5. Load the droplet reader plate holder containing the sample plate into the reader chamber and locate it securely onto the magnetic base.
6. Press the Open/Close button on the lid of the droplet reader to close it.
7. Open the analysis software interface, select the Add Plate tab and prepare a new sample plate as follows:
   1. Click Add Plate and then Configure Plate. 
   2. In the Plate Information tab enter a Plate Name, select Supermix for Probes (no dUTP) from the Supermix drop-down menu and enter a file name under Save Data File As.
   3. In the Well Selection tab highlight the wells to be analyzed and click Include Selected Wells.
      NOTE: Steps 6.7.4 – 6.7.7 are optional.
   4. In the Well Information tab, select the wells to be annotated.
      NOTE: Refer to Figure 1D for an example of the Well Information tab interface.
   5. Select Direct Quantification (DQ) from the Experiment Type drop-down menu, populate the Sample Description, Sample Type & Target Name boxes, and add Well Notes and/or Plate Notes as required.
   6. Click Apply. The information in the fields will be applied to the selected wells.
   7. Repeat steps 6.7.4–6.7.6 until all sample wells have been annotated. 
8. Once the plate configuration is complete, click Start Run.
9. When the run is complete, discard the empty sample plate and empty the droplet reader waste bottle if necessary. 
10. Proceed to Step 7.

7. Analysis of results

NOTE: The droplet reader measures the fluorescence intensity in the FAM and HEX channel for each droplet in a sample. In a successful assay, droplets fall into one of two categories for each probe: Negative (meaning the target was not present in the droplet) or Positive (meaning the target was present in the droplet). Prior to analyzing samples, ensure that each well contains >10,000 droplets and has two clearly separate populations of low fluorescence (negative) and high fluorescence (positive) droplets in each channel (Figure 2A).

1. Select the Data Analysis tab and open the file to be analyzed from the My Datafiles menu.
2. Click the 1D Amplitude tab for single-color experiments or the 2D Amplitude tab for two-color experiments.
3. Apply a fluorescence threshold to each channel to differentiate the positive and negative droplets. The analysis software will attempt to apply an automatic threshold on a sample-by-sample and channel-by-channel basis, but if this appears to have failed, or looks inaccurate, then manually apply the threshold as follows:
   1. Select the wells that require thresholding.
   2. Manually apply the threshold in the clear space between the positive and negative populations on the amplitude plot using the Threshold Line Mode tool. When setting manual thresholds in 2D view, ensure that the crosshair is placed so that the four droplet populations (double negative, single positive FAM, single positive HEX, and double positive) are clearly separated.
      NOTE: Thresholding can either be done well-by-well or applied to multiple wells at once.
4. Once all samples have had a threshold applied, export the results as a .csv file for further analysis by clicking on the Data Table tab, clicking Import/Export and selecting Export Visible Data to CSV. Enter a suitable file name and click Save.
5. Calculate the mtDNA copy number in the initial sample as follows:
   Copy Number = mtDNA target concentration × 22 × 1/fraction of total lysate input
   NOTE: If two mitochondrial probes are used, then the concentration of the two targets is averaged before making the calculation. For samples containing more than one cell, the copy number per cell is calculated by dividing by the number of cells in the sample. It is important to use the 'Concentration' value (i.e., number of copies per microliter) multiplied by the initial sample volume of 22 µL, rather than the 'Copies per 20 µL' value calculated in the .csv file, since the mtDNA copies left in the 2 µL overage required by the droplet generator must be taken into account when calculating the absolute copy number of the initial sample.
6. For cells carrying heteroplasmic mtDNA deletions, calculate the copy number using the results from the mtDNA target outside the deleted region (i.e., the target present in both deleted and WT mtDNA molecules). The deletion heteroplasmy is calculated using the concentrations of the target inside the deleted region and the target outside the deleted region as follows:
   

Results

Following droplet generation, a clear layer of opaque droplets is visible floating on top of the oil phase in each well (Figure 1B). Droplet formation can be adversely affected by the presence of detergents in the input lysate when performing experiments on single cells. Using the lysis protocol described in 2.1.2., droplet yields above the recommended level of 10,000 are routinely achieved, despite the presence of a small residual amount of TWEEN-20 in the final sample (

Discussion

The protocol described here is applicable across a wide range of cell types and species in addition to those discussed above, although careful optimization of new assay designs will be key to ensure that accuracy and repeatability of the method are maintained when moving away from previously validated primer/probe combinations. When working with single cells it is vital to ensure that sample collection is performed as accurately as possible (e.g., using stringent single-cell parameters when sorting cells by FACS) to ensu...

Materials

Name	Company	Catalog Number	Comments
50% Tween-20 solution	Novex	3005
Automated droplet-generating oil	Bio Rad	1864110	Commercial oil formulation used to generate the oil/droplet emulsion (used in Protocol Step 4.1)
C1000 PCR machine with deep-well block	Bio Rad	1851197	PCR thermocycler equipped with a deep-well heating block, used for cell lysis (Protocol Step 2.1.2.) and PCR cycling (Protocol Step 5)
Collection plate cooling block	Bio Rad	12002819	Cooling block that keeps samples chilled during droplet generation (used in Protocol step 4.3)
ddPCR 96-well plates	Bio Rad	12001925	96-well plates pipet tips designed for use in the QX200 AutoDG droplet generator, used for sample preparation (Protocol step 3.4) and droplet collection (Protocol step 4.3)
ddPCR droplet reader oil	Bio Rad	1863004	Commercial oil formulation used by the droplet reader (used in Protocol step 6.1)
ddPCR Supermix for Probes (no dUTP)	Bio Rad	1863023	Commercial supermix for use in ddPCR experiments utilising probes (used in Protocol Step 3.3)
DG32 automated droplet generator cartridges	Bio Rad	1864108	Microfluidic cartridges used in the QX200 AutoDG droplet generator to generate the oil/droplet emulsion (used in Protocol Step 4.3)
Fetal bovine serum	Gibco	10270-106	Qualified fetal bovine serum
Foil plate covers	Bio Rad	1814040	Foil plate covers used to seal droplet collection plates after droplet generation (used in Protocol step 4.6)
HEK 293T cells	Takara	632180	Commercial subclone of the transformed human embryonic kidney cell line, HEK 293, expressing the SV40 Large-T antigen
HeLa cells	ECACC	93021013	Human cervix epitheloid carcinoma cells
High glucose DMEM	Gibco	13345364	4.5g/L D-Glucose, with L-glutamine and sodium pyruvate
Human cybrids	University of Miami
Mouse embryonic fibroblasts	Newcastle University		Immortalized from C57Bl/6 mice
Nuclease-free water	Ambion	AM9937
PCR plate seals	Pierce	SP-0027	Clear adhesive plate seals, only used pre-droplet generation (foil seal must be used in step 4.6)
Pipet Tip Waste Bins	Bio Rad	1864125	Disposable collection bin used to collect discarded tips in the QX200 AutoDG droplet generator (used in Protocol step 4.3)
Pipet tips for AutoDG system	Bio Rad	1864120	Filtered pipet tips designed for use in the QX200 AutoDG droplet generator (used in Protocol step 4.3)
Primary human dermal fibroblast cells	Newcastle Biobank
Primers/Probes	IDT	N/A	Exact primer/probe sequences will be assay dependent. Primers and probes used in this study are given in Table 1
Proteinase K 20 mg/mL solution	Ambion	AM2546
PX1 PCR plate sealer	Bio Rad	1814000	Applies foil seals to ddPCR sample plates after droplet generation (used in Protocol Step 4.6)
QX Manager software	Bio Rad	12012172	Droplet reader set up & analysis software (used in Protocol Steps 6 & 7)
QX200 AutoDG droplet generator	Bio Rad	1864101	Automated microfluidic droplet generator (used in Protocol Step 4)
QX200 droplet reader	Bio Rad	1864003	Droplet reader (used in Protocol Step 6)
Trizma pre-set crystals pH 8.3	Sigma	T8943-100G